The major focus of this project is to investigate the molecular biology and biochemistry of the ras oncogenes and the ras p21 proteins. The long-range objective is to elucidate molecular mechanisms of cell transformation induced by these genes and their products. We have found novel phosphorylation of p21 of H- and K-ras genes, distinct from autophosphorylation previously known in Harvey and Kirsten viral ras oncogene products. The phosphorylation of ras protein in cells was stimulated by phorbol ester. Protein kinase C phosphorylated p21 in vitro. The present results suggest that these novel phosphorylations were mediated by kinase C. Structure-function of ras proteins were investigated by methods of site-directed mutagenesis, enzymology, and immunochemistry. Results indicate that the structure of the GTP-binding domain of p21 is very similar to that of a super family of G-proteins important in cellular signal transduction. The GTP-binding domain functions as a switch region for the regulatory roles of p21. Classes of mutants have been found that either render p21 in a permanent on mode, or inactivate p21. Studies on a neutralizing monoclonal antibody, which has been shown to block p21 cellular activities, indicates that the dissociation off-rate of prebound GDP for the exchange with GTP is important for p21 function. The nucleotide exchange rate is significantly higher in the viral ras p21 than that of the proto- oncogene p21, suggesting its role in high oncogenicity of viral oncogenes.